PROJECT SUMMARY Atherosclerosis is a disease of the blood vessel wall that causes heart attacks and strokes. Despite major advances in medical and surgical management, atherosclerosis still causes significant morbidity and mortality. For example, drug therapy that dramatically lowers LDL-cholesterol (to ~30 mg/dL) only marginally decreases adverse cardiovascular events and mortality. New approaches, beyond LDL lowering, are needed. The broad, long-term objective of this project is to develop a therapy that prevents and reverses atherosclerosis via interventions targeted at the blood vessel wall. This novel therapy involves the introduction and expression of disease-preventing genes in the cells that line blood vessel walls and is accordingly termed ?atheroprotective gene therapy.? Blood vessels treated with this gene therapy would not develop atherosclerosis because they are genetically modified to resist the underlying biological processes that cause atherosclerosis: accumulation of cholesterol and inflammatory cells and activation of vascular cell inflammatory pathways. This project is focused on gene therapy that prevents cholesterol accumulation (and associated inflammation) in blood vessel walls. Vessel wall-targeted gene therapy is particularly well suited for prevention of atherosclerosis in veins used for coronary artery bypass grafts. Vein-graft atherosclerosis progresses rapidly, leads to graft narrowing and occlusion, and is inadequately treated. Our approach could eventually eliminate vein-graft atherosclerosis. There are 3 specific aims, all of which are carried out in rabbits. The aims are focused on developing clinically useful atheroprotective gene therapy, delivered by a promising gene-transfer vector, ?helper-dependent adenovirus? (HDAd). HDAd is an attractive vector for human gene therapy because it expresses therapeutic genes stably for years in animals (including nonhuman primates) and is relatively non-inflammatory. The 3 specific aims exploit the promise of HDAd and take the next critical steps towards developing gene therapy that prevents and reverses atherosclerosis: Aim 1 will test whether HDAd-mediated expression of apolipoprotein A-I (apoA-I) or ATP-binding cassette subfamily A, member 1 (ABCA1) can prevent atherosclerosis in grafted rabbit veins. Aim 2 will develop novel expression cassettes that achieve high-level, stable, cell-specific transgene expression in endothelium in vivo. Aim 3 will identify the mechanisms through which atheroprotective gene therapy that produces apoA-I from endothelial cells prevents atherosclerosis. Accomplishment of the 3 aims will bring clinical vascular gene therapy closer to implementation by testing 2 promising therapeutic genes in a large animal model (Aim 1). Accomplishment of the aims may also yield novel vector platforms that are useful for expressing transgenes at high levels in endothelial cells and may also provide insights into mechanisms of endothelial cell gene transcription (Aim 2). Identification of the mechanisms through which apoA-I vascular gene therapy prevents atherosclerosis (Aim 3) will suggest approaches for improving apoA-I vascular gene therapy and for the development of adjunctive gene therapies.